Essential thrombocythemia (ET) and primary myelofibrosis (PMF) are chronic diseases characterized by clonal hematopoiesis and hyperproliferation of terminally differentiated myeloid cells. The disease is driven by somatic mutations in exon 9 of CALR or exon 10 of MPL or JAK2-V617F in >90% of the cases, whereas the remaining cases are termed "triple negative." We aimed to identify the disease-causing mutations in the triple-negative cases of ET and PMF by applying whole-exome sequencing (WES) on paired tumor and control samples from 8 patients. We found evidence of clonal hematopoiesis in 5 of 8 studied cases based on clonality analysis and presence of somatic genetic aberrations. WES identified somatic mutations in 3 of 8 cases. We did not detect any novel recurrent somatic mutations. In 3 patients with clonal hematopoiesis analyzed by WES, we identified a somatic MPL-S204P, a germline MPL-V285E mutation, and a germline JAK2-G571S variant. We performed Sanger sequencing of the entire coding region of MPL in 62, and of JAK2 in 49 additional triple-negative cases of ET or PMF. New somatic (T119I, S204F, E230G, Y591D) and 1 germline (R321W) MPL mutation were detected. All of the identified MPL mutations were gain-of-function when analyzed in functional assays. JAK2 variants were identified in 5 of 57 triple-negative cases analyzed by WES and Sanger sequencing combined. We could demonstrate that JAK2-V625F and JAK2-F556V are gain-of-function mutations. Our results suggest that triple-negative cases of ET and PMF do not represent a homogenous disease entity. Cases with polyclonal hematopoiesis might represent hereditary disorders.

CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences Vienna Austria;

CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences Vienna Austria; Center of Molecular Biology and Gene Therapy Department of Internal Medicine Hematology and Oncology University Hospital Brno and Medical Faculty Masaryk University Brno Czech Republic; and

CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences Vienna Austria; Department of Internal Medicine 1 Division of Hematology and Blood Coagulation Medical University of Vienna Vienna Austria;

Department of Hematology Oncology Fondazione Istituto di Ricovero e Cura a Carattere Scientifico Policlinico San Matteo Pavia Italy;

Department of Hematology Oncology Fondazione Istituto di Ricovero e Cura a Carattere Scientifico Policlinico San Matteo Pavia Italy; Department of Molecular Medicine University of Pavia Pavia Italy

Department of Internal Medicine 1 Division of Hematology and Blood Coagulation Medical University of Vienna Vienna Austria;

Ludwig Institute for Cancer Research Brussels Belgium; de Duve Institute Université Catholique de Louvain Brussels Belgium;

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A novel germline hyperactivating JAK2 mutation L604F